PiggyBac-Engineered T Cells Expressing CD19-Specific CARs that Lack IgG1 Fc Spacers Have Potent Activity against B-ALL Xenografts

David C Bishop; Ning Xu; Benjamin Tse; Tracey A O'Brien; David J Gottlieb; Alla Dolnikov; Kenneth P Micklethwaite

doi:10.1016/j.ymthe.2018.05.007

PiggyBac-Engineered T Cells Expressing CD19-Specific CARs that Lack IgG1 Fc Spacers Have Potent Activity against B-ALL Xenografts

Mol Ther. 2018 Aug 1;26(8):1883-1895. doi: 10.1016/j.ymthe.2018.05.007. Epub 2018 Jun 1.

Authors

David C Bishop¹, Ning Xu², Benjamin Tse³, Tracey A O'Brien⁴, David J Gottlieb⁵, Alla Dolnikov⁴, Kenneth P Micklethwaite⁶

Affiliations

¹ Westmead Institute for Medical Research, Sydney, NSW, Australia; Department of Haematology, Westmead Hospital, Sydney, NSW, Australia; Blood and Bone Marrow Transplant Unit, Westmead Hospital, Sydney, NSW, Australia; Sydney Medical School, The University of Sydney, Sydney, NSW, Australia.
² Blood & Marrow Transplant Facility, Kids Cancer Centre, Sydney Children's Hospital, Sydney, NSW, Australia; Children's Cancer Institute, University of New South Wales, Sydney, NSW, Australia.
³ Children's Cancer Institute, University of New South Wales, Sydney, NSW, Australia; Faculty of Medicine, University of New South Wales, Sydney, NSW, Australia.
⁴ Blood & Marrow Transplant Facility, Kids Cancer Centre, Sydney Children's Hospital, Sydney, NSW, Australia; Children's Cancer Institute, University of New South Wales, Sydney, NSW, Australia; Faculty of Medicine, University of New South Wales, Sydney, NSW, Australia.
⁵ Westmead Institute for Medical Research, Sydney, NSW, Australia; Department of Haematology, Westmead Hospital, Sydney, NSW, Australia; Blood and Bone Marrow Transplant Unit, Westmead Hospital, Sydney, NSW, Australia; Sydney Medical School, The University of Sydney, Sydney, NSW, Australia; Sydney Cellular Therapies Laboratory, Westmead Hospital, Sydney, NSW, Australia; Department of Medicine, Westmead Hospital, Sydney, NSW, Australia.
⁶ Westmead Institute for Medical Research, Sydney, NSW, Australia; Department of Haematology, Westmead Hospital, Sydney, NSW, Australia; Blood and Bone Marrow Transplant Unit, Westmead Hospital, Sydney, NSW, Australia; Sydney Medical School, The University of Sydney, Sydney, NSW, Australia; Sydney Cellular Therapies Laboratory, Westmead Hospital, Sydney, NSW, Australia. Electronic address: kenneth.micklethwaite@sydney.edu.au.

Abstract

Clinical trials of CD19-specific chimeric antigen receptor (CAR19) T cells have demonstrated remarkable efficacy against relapsed and refractory B cell malignancies. The piggyBac transposon system offers a less complex and more economical means for generating CAR19 T cells compared to viral vectors. We have previously optimized a protocol for the generation of CAR19 T cells using the piggyBac system, but we found that CAR19 T cells had poor in vivo efficacy and persistence, probably due to deleterious FcγR interactions with the CAR's IgG1 Fc-containing spacer domain. We therefore designed three CD19-specifc CARs that lacked the IgG1 Fc region, and we incorporated combinations of CD28 or 4-1BB transmembrane and co-stimulatory domains. PiggyBac-generated CAR19 T cells expressing these re-designed constructs all demonstrated reactivity in vitro specifically against CD19⁺ cell lines. However, those combining CD28 transmembrane and co-stimulatory domains showed CD4 predominance and inferior cytotoxicity. At high doses, CAR19 T cells were effective against B-ALL in a xenograft mouse model, regardless of co-stimulatory domain. At diminishing doses, 4-1BB co-stimulation led to greater potency and persistence of CAR19 T cells, and it provided protection against B-ALL re-challenge. Production of potent CAR T cells using piggyBac is simple and cost-effective, and it may enable wider access to CAR T cell therapy.

Keywords: B cell malignancy; CAR T cell; CD19; acute lymphoblastic leukemia; cellular immunotherapy; chimeric antigen receptor; murine xenograft model; non-viral vector; piggyBac transposase; transposon.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
Cell Line, Tumor
DNA Transposable Elements
Humans
Immunotherapy, Adoptive / methods
Jurkat Cells
K562 Cells
Mice
Precursor B-Cell Lymphoblastic Leukemia-Lymphoma / genetics
Precursor B-Cell Lymphoblastic Leukemia-Lymphoma / immunology
Precursor B-Cell Lymphoblastic Leukemia-Lymphoma / therapy*
Receptors, Antigen, T-Cell / metabolism*
Receptors, IgG / genetics*
T-Lymphocytes / transplantation*
Treatment Outcome
Xenograft Model Antitumor Assays

Substances

CD19-specific chimeric antigen receptor
DNA Transposable Elements
Receptors, Antigen, T-Cell
Receptors, IgG